HIV-1 persists in CD4⁺ T cells and brain microglia through host factors that enforce viral latency, yet the mechanisms that stabilize key transcriptional regulators remain incompletely understood. Here, we identify the YEATS domain-containing protein ENL and its associated deubiquitinase USP7 as a host complex that maintains HIV-1 latency. USP7 stabilizes BRD4 by deubiquitination, suppressing HIV transcription and sustaining viral quiescence. Disruption of the ENL-USP7 complex using selective PROTACs reactivates latent HIV in cell line models, as well as in resting CD4⁺ T cells and microglia isolated from people with HIV on antiretroviral therapy. These findings uncover a critical ENL-USP7-BRD4 axis that enforces HIV-1 latency and highlight USP7 as a potential target for latency-reversing strategies. Highlights:ENL, a YEATS domain-containing crotonylation reader, acts as a suppressor rather than an activator of HIV-1 transcription.ENL recruits USP7 to stabilize BRD4 and enforce viral latency.Disruption of the ENL-USP7-BRD4 axis reactivates latent HIV in T cells and microglia.Targeting USP7 or ENL reveals a therapeutic vulnerability in HIV reservoirs.

The estrogen receptor (ER) is hypothesized to directly influence HIV transcription and latency but is also critical for immune signaling. However, the mechanisms of action of the ER in immune cells in the context of HIV are limited, and relevant to HIV cure strategies, the influence of latency reversal agents (LRAs) on the ER pathway are unknown. We evaluated (a) the effect of estrogen (E2) on the nuclear translocation of ERα in CD4+ T cells; (b) the ability of Fulvestrant, a selective estrogen receptor degrader (SERD), and ARV-471, a potent, PROteolysis TArgeting Chimera (PROTAC) selective ERα degrader to modulate ERα; and c) the effect of different classes of LRAs on ERα signaling. In contrast to what has been demonstrated in oncology, E2 did not induce ERα nuclear translocation in CD4+ T cells. Similarly, neither Fulvestrant nor ARV-471 induced degradation of ERα in CD4+ T cells. LRAs significantly downregulated ERα gene and protein expression in both PBMCs and CD4+ T cells. Collectively, our results suggest that estrogen influences on HIV transcription are not likely a consequence of canonical nuclear ERα mechanisms. The consequences of LRA downregulation of ERα, a protein important for immune signaling, warrant further investigation.

As cannabis use continues to rise among people with HIV (PWH), understanding its impact on immune function in this population is becoming increasingly important. To provide new insights on how cannabis modulates immune function, we analyzed single-nucleus multi-omic profiles of peripheral blood mononuclear cells (PBMCs) from PWH to characterize the changes in gene expression and chromatin accessibility associated with chronic cannabis exposure. We identified numerous differentially expressed genes (DEGs) between cannabis users and non-users in each cell type, approximately half of which were unique to individual cell types. Changes in pro- and anti-inflammatory gene expression associated with cannabis use are dependent on cell lineage and type. We identified hundreds of differential chromatin accessibility regions in each cell type, including cis-regulatory elements correlated with cell-type-dependent DEGs (e.g. NFKBIA in CD4+ T cells and CCL3L1 in classical monocytes). Multiple cannabis-associated transcription factors (e.g., NFKB1, FOS, and TCF7) emerge as regulators of the differentially expressed inflammatory genes. Furthermore, cannabis altered the communication between classical monocytes and lymphocytes. These findings indicate that cannabis-induced immunomodulatory effects are profound, dynamic and complex among cell types and that transcriptional changes are regulated at least in part by epigenetic mechanisms.

The role of epigenetic regulation in HIV latency remains incompletely understood. We show that histone deacetylase 3 (HDAC3) inhibits trans-activator of transcription (Tat)-mediated HIV transcription through histone decrotonylation (HDCR), independent of deacetylase activity. Chemical biology approaches identified selective HDCR inhibitors (HDCRis) that reverse HIV latency with minimal impact on other histone acylations. Although HDAC2, HDAC3, and HDAC8 exhibit HDCR activity, genetic and chemical studies reveal that the HDCRi citarinostat is selective for HDAC3 and HDAC8. Molecular docking suggests that HDCRi binds outside the zinc-binding pocket, distinct from the classical HDAC inhibitor vorinostat (SAHA, suberoylanilide hydroxamic acid). Key residues (arginine-265, arginine-301, glutamine-113, and aspartic acid-57) are essential for HDCR selectivity, as their mutation abolishes HDCR activity and increases histone crotonylation without altering other acylation marks. Citarinostat increases histone crotonylation at the HIV long terminal repeat, robustly activating HIV transcription in cell lines, primary CD4+ T cells, and brain microglia from simian immunodeficiency virus-infected nonhuman primates and participants enrolled in the Last Gift rapid research autopsy cohort, highlighting HDCR as a promising therapeutic target for HIV latency.

Immunological mechanisms regulating HIV rebound after antiretroviral therapy (ART) interruption remain unclear. We examined relationships between host factors, HIV reservoir, and HIV time-to-rebound after analytical treatment interruption (ATI) by characterizing pre-ATI peripheral blood mononuclear cells (PBMCs) from 75 ART-suppressed people with HIV (PWH) using high-parameter methods. Across interventional (CLEAR, TEACH, and REDUC) and non-interventional (A5345) cohorts, delayed rebound was not associated with intact HIV. Cohort-specific immune effectors were associated with delayed rebound. RNA sequencing of CD4+ T cells from A5345 revealed that the mTOR inhibitor DDIT4 and zinc finger protein ZNF254 were associated with delayed rebound. In vitro and in vivo studies demonstrated that DDIT4 and ZNF254 suppressed HIV expression. Metformin induced DDIT4 and suppressed HIV expression in primary cells and cells from ART-suppressed PWH, suggesting that this affordable diabetes drug could be repurposed to silence HIV. Our results support the pursuit of both immune- and HIV-silencing strategies to achieve ART-free HIV remission.

ABSTRACT HIV cure strategies that aim to induce viral reactivation for immune clearance leverage latency reversal agents to modulate host pathways which directly or indirectly facilitate viral reactivation. Inhibition of bromo and extra-terminal domain (BET) family member BRD4 reverses HIV latency, but enthusiasm for the use of BET inhibitors in HIV cure studies is tempered by concerns over inhibition of other BET family members and dose-limiting toxicities in oncology trials. Here, we evaluated the potential for bivalent chemical degraders targeted to the BET family as alternative latency reversal agents. We observed that despite highly potent and selective BRD4 degradation in primary CD4+ T-cells from ART-suppressed donors, BRD4 degraders failed to induce latency reversal as compared to BET inhibitors. Furthermore, BRD4 degraders failed to mimic previously observed synergistic HIV reactivation between BET inhibitors and an activator of the non-canonical NF-κB pathway. Mechanistic investigation of this discrepancy revealed that latency reversal by BET inhibitors is not related to the abatement of competition between Tat and BRD4 for P-TEFb, but rather the ability of BRD4 to disrupt 7SK and increase the levels of free P-TEFb. This activity is dependent on the shift of BRD4 from chromatin-bound to soluble and retargeting of P-TEFb to chromatin, which is dependent on intact BRD4 but independent of the bromodomains. IMPORTANCE Multiple factors and pathways contribute to the maintenance of HIV latency, including bromo and extra-terminal domain (BET) family member BRD4. While small molecule inhibitors of the BET family result in latency reversal, enthusiasm for the use of BET inhibitors in HIV cure is limited due to toxicity concerns. We examined BRD4-selective chemical degraders as alternatives to BET inhibitors but found two robust degraders failed to induce latency reversal. We observed key differences in the ability of BET inhibitors versus BET degraders to disrupt P-TEFb, a key cellular activator of transcription and a complex required for HIV reactivation. We present a new model for the role of BRD4 in HIV latency and propose that BRD4 be reconsidered as an activator rather than a repressor of HIV transcription in the context of HIV cure strategies.

Human Immunodeficiency virus (HIV) infection is regulated by a wide array of host cell factors that combine to influence viral transcription and latency. To understand the complex relationship between the host cell and HIV-1 latency, we performed a lentiviral CRISPR screen that targeted a set of host cell genes whose expression or activity correlates with HIV-1 expression. We further investigated one of the identified factors - the transcription factor ETS1, and found that it is required for maintenance of HIV-1 latency in both latently infected cell lines and in a primary CD4 T cell latency model. Interestingly, ETS1 played divergent roles in actively infected and latently infected CD4 T cells, with knockout of ETS1 leading to reduced HIV-1 expression in actively infected cells, but increased HIV-1 expression in latently infected cells, indicating that ETS1 can play both a positive and negative role in HIV-1 expression. CRISPR/Cas9 knockout of ETS1 in CD4 T cells from ART-suppressed people with HIV-1 (PWH) confirmed that ETS1 maintains transcriptional repression of the clinical HIV-1 reservoir. Transcriptomic profiling of ETS1-depleted cells from PWH identified a set of host cell pathways involved in viral transcription that are controlled by ETS1 in resting CD4 T cells. In particular, we observed that ETS1 knockout increased expression of the long non-coding RNA MALAT1 that has been previously identified as a positive regulator of HIV-1 expression. Furthermore, the impact of ETS1 depletion on HIV-1 expression in latently infected cells was partially dependent on MALAT1. Additionally, we demonstrate that ETS1 knockout resulted in enhanced abundance of activating modifications (H3K9Ac, H3K27Ac, H3K4me3) on histones located at the HIV-1 long terminal repeat (LTR), indicating that ETS1 regulates the activity of chromatin-targeting complexes at the HIV-1 LTR. Overall, these data demonstrate that ETS1 is an important regulator of HIV-1 latency that impacts HIV-1 expression through repressing MALAT1 expression and by regulating modification of proviral histones.

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing toward inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells of people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo, and we detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout, suggesting that INTS12 prevents full-length HIV RNA production in primary T cells. Finally, we found that INTS12 more generally limits the efficacy of a variety of LRAs with different mechanisms of action.

The 38th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), took place March 17-21, 2025 in Las Vegas, Nevada, USA. The annual meeting brought together leading scientists from across academia, industry, and government to present the latest advances in antiviral research. Topics included discovery and development of novel antiviral agents, innovative therapeutic approaches, vaccine technologies, host-targeted strategies, and responses to emerging and re-emerging viral threats. ICAR 2025 featured keynote talks, short oral presentations, poster sessions and special sessions to encourage discussions between attendees and foster interdisciplinary collaboration. Several events were held to support the next generation of antiviral researchers, including dedicated sessions and networking opportunities focused on mentorship and career development for students, postdoctoral fellows, and early-career scientists. Importantly, ISAR continues to serve as a cornerstone for international collaboration and innovation in antiviral science, and the society is eager to continue these efforts at the 39th ICAR, to be held in Prague, Czech Republic, from April 27-May 1, 2026.

Human gastrointestinal (GI) tissues are a major site of HIV-1 viral persistence, but the nature of the GI reservoir remains poorly described. To characterize the GI HIV reservoir, we profiled cells from GI tissue and matched PBMCs from 10 people with HIV on antiretroviral therapy using single-cell RNA sequencing. We identified distinct compartment-specific patterns of gene expression, highlighting key differences between blood and colon CD4+ T cell populations. vRNA+ cells from both blood and GI tissue were heterogeneous and found in multiple subtypes of CD4+ T cells, although vRNA+ cells were particularly enriched in cells with Th17 or Treg17 phenotypes. Transcriptomic comparison of HIV vRNA+ and vRNA- T cells revealed 116 differentially expressed genes that were associated with HIV infection, including ZBED2, MAF, and IL17F. These data provide what we believe to be new information regarding the GI-resident HIV reservoir and suggest that compartment-specific patterns of gene expression are associated with HIV infection.

Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, the within-host evolutionary origins of hypermutated sequences are incompletely understood because phylogenetic inference algorithms, which assume that mutations gradually accumulate over generations, incorrectly reconstruct their ancestor-descendant relationships. Using >1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up − including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and >500 proviruses isolated over a median 9 years on ART − we evaluated three approaches for removing hypermutation from nucleotide alignments. Our goals were to 1) reconstruct accurate phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the tested approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env-intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env-intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, indicating that the corrected trees can be used for molecular dating. Use of these trees to infer the integration dates of hypermutated proviruses persisting during ART revealed that these spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env-intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories, allow insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

BACKGROUND:The HIV-1 reservoir in CD4+ T cells (HRCD4) pose a major challenge to curing HIV, with many of its mechanisms still unclear. HIV-1 DNA integration and immune responses may alter the host's epigenetic landscape, potentially silencing HIV-1 replication. METHODS:This study used bisulphite capture DNA methylation sequencing in CD4+ T cells from the blood of 427 virally suppressed women with HIV to identify differentially methylated sites and regions associated with HRCD4. RESULTS:The average total HRCD4 size was 1409 copies per million cells, with most proviruses defective and only a small proportion intact. The study identified 245 differentially methylated CpG sites and 85 regions linked to HRCD4 size, with 52% of significant sites in intronic regions. Genes associated with HRCD4 were involved in viral replication, HIV-1 latency and cell growth and apoptosis. HRCD4 size was inversely related to DNA methylation of interferon signalling genes and positively associated with methylation at known HIV-1 integration sites. HRCD4-associated genes were enriched on the pathways related to immune defence, transcription repression and host-virus interactions. CONCLUSIONS:These findings suggest that HIV-1 reservoir is linked to aberrant DNA methylation in CD4+ T cells, offering new insights into epigenetic mechanisms of HIV-1 latency and potential molecular targets for eradication strategies. KEY POINTS:Study involved 427 women with HIV. Identified 245 aberrant DNA methylation sites and 85 methylation regions in CD4+ T cells linked to the HIV-1 reservoir. Highlighted genes are involved in viral replication, immune defence, and host genome integration. Findings suggest potential molecular targets for eradication strategies.

PURPOSE OF REVIEW:As more women with HIV survive into older age, the menopausal transition has emerged as a critical yet underexplored determinant in HIV pathogenesis. Declining exposure to estrogens during menopause alters innate and adaptive immunity, driving inflammation, comorbidities, and viral persistence. RECENT FINDINGS:Estrogen influences both innate and adaptive immune responses. Estradiol enhances plasmacytoid dendritic cell type I interferon (IFN) production through Toll-like Receptor 7 (TLR7), promotes natural killer (NK) cell activity, and tempers monocyte/macrophage activation. Menopause reverses these effects, contributing to elevated inflammatory mediators. On the adaptive side, estrogen loss increases T-cell activation and exhaustion, impairs B-cell responses, and removes estrogen receptor (ER)-mediated suppression of HIV transcription. Together, these shifts may promote stabilization or expansion of the HIV reservoir in perimenopausal women with HIV, in contrast to the gradual decay often observed in men on antiretroviral therapy (ART). SUMMARY:Estrogen depletion during menopause reshapes immunity in women with HIV, fueling chronic inflammation, comorbidity risk, and HIV reservoir persistence. Integrating reproductive aging into HIV cure and comorbidity research, and testing hormone-based and anti-inflammatory interventions, will be essential to improve health outcomes for aging women with HIV.

Background:We evaluated rapid start of integrase-based antiretroviral therapy (ART) during acute HIV-1. Methods:Adult participants initiated co-formulated dolutegravir/abacavir/lamivudine within 30 days of acute HIV-1 diagnosis. HLA-B*57-positive participants were excluded by rapid, flow cytometry screening. We evaluated HIV-1 RNA levels, CD4+ T-cell subsets, and change in replication competent HIV-1. Results:Forty adults screened with 3 excluded due to positive HLA-B*57:01 or hepatitis B surface antigen results. All 37 participants starting study treatment suppressed to <200 copies/mL by week 24 (median of 4 weeks, interquartile range 3.4-5.1); 86% and 95% were <50 copies/mL at weeks 48 and 96, respectively. We observed a median 2.4-fold decline in frequency of resting CD4+ T-cell infection in a subset of participants providing 96 week samples. ART in acute HIV-1 resulted in CD4+ T-cell memory subpopulations similar to people without HIV-1 and preserved CD4+ and CD8+ T-cell frequencies compared to people starting ART in chronic HIV. Thirty-four participants required rapid HLA-B*57 testing at screening; 97% resulted ≤24 hours, and 71% started ART ≤24 hours. Conclusions:Integrase-based ART during acute HIV-1 resulted in brisk viral suppression, preservation of CD4+ T-cell subsets, and decline in resting CD4+ T-cell infection.

Initial efforts to control HIV infection include an autologous neutralizing antibody (aNAb) response. aNAbs bind Env trimers of the infecting HIV strain to neutralize virus but are not very effective at controlling HIV, as the virus quickly develops escape mutations to evade neutralization. Nevertheless, recent evidence suggests that aNAbs exert ongoing immune pressure on viral isolates in people living with HIV (PWH) treated with anti-retroviral therapy (ART) during chronic and early infection. In this issue of the JCI, McMyn et al. studied the dynamics of aNAb resistance in a cohort of 31 PWH treated with ART. Notably, a large proportion of HIV reservoir viral isolates were resistant to aNAb neutralization, which correlated with longer duration on uninterrupted ART, suggesting that selection for aNAb-resistant isolates occurs as reservoir cells containing neutralization-sensitive isolates are eliminated. aNAb resistance was not attributed to waning antibody response, which persisted for over 20 years despite viral suppression.

Background The histone deacetylase inhibitor vorinostat (VOR) can reverse human immunodeficiency virus type 1 (HIV-1) latency in vivo and allow T cells to clear infected cells in vitro. HIV-specific T cells (HXTCs) can be expanded ex vivo and have been safely administered to people with HIV (PWH) on antiretroviral therapy.Methods Six PWH received infusions of 2 x 107 HXTCs/m(2) with VOR 400 mg, and 3 PWH received infusions of 10 x 107 HXTCs/m(2) with VOR. The frequency of persistent HIV by multiple assays including quantitative viral outgrowth assay (QVOA) of resting CD4+ T cells was measured before and after study therapy.Results VOR and HXTCs were safe, and biomarkers of serial VOR effect were detected, but enhanced antiviral activity in circulating cells was not evident. After 2 x 107 HXTCs/m(2) with VOR, 1 of 6 PWH exhibited a decrease in QVOA, and all 3 PWH exhibited such declines after 10 x 107 HXTCs/m(2) and VOR. However, most declines did not exceed the 6-fold threshold needed to definitively attribute decline to the study intervention.Conclusions These modest effects provide support for the strategy of HIV latency reversal and reservoir clearance, but more effective interventions are needed to yield the profound depletion of persistent HIV likely to yield clinical benefit. Clinical Trials Registration. NCT03212989.Conclusions These modest effects provide support for the strategy of HIV latency reversal and reservoir clearance, but more effective interventions are needed to yield the profound depletion of persistent HIV likely to yield clinical benefit. Clinical Trials Registration. NCT03212989. The HIV latency reversal agent vorinostat was well tolerated when given with expanded, autologous, HIV-specific cytotoxic T cells, but only modest effects were seen on the pool of latently infected cells that persist in people with HIV on therapy.